Viral vector stabilization

ABSTRACT

Combining viral vector with surfactant preserves vector infectivity, and surfactant provided an unexpected benefit by protecting viral vector from damage due to transient elevated temperature.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. Ser. No. 15/781,707filed 6 Jun. 2018, which is a United States National Stage entry of andasserts priority to PCT/US2017/025727 filed 3 Apr. 2017, which in turnasserts priority from United States provisional patent filing Ser. No.62/322,452, filed 14 Apr. 2016, the contents of which are hereincorporated by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

None

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not applicable.

SEQUENCE LISTING

None.

PRIOR DISCLOSURES BY AN INVENTOR

None.

BACKGROUND

Infective viral particles are useful for many medical uses. For example,attenuated virus are useful as vaccines. Similarly, infective (yet oftenreplication deficient) virus are useful as gene therapy vectors todeliver therapeutic transgenes.

The art teaches that infective virus particles are less than perfectlyefficient in transfecting host cells. The art teaches that transfectionefficiency may be increased by pre-treating the host cells withsurfactant.

The art, however, also cautions that surfactant may damage viralparticles, and thus reduce their infectivity. The art thus teaches topre-treat host cells with surfactant immediately before viral vectortransfection, yet avoid mixing the viral vector and surfactant andstoring for any significant time. The art thus teaches to provide vectorand surfactant as physically separate components for therapy.

We thus attempted to quantify the deleterious effect of surfactant onviral vector. To do so, we prepared a viral vector, measured itsinfectivity, mixed samples of it with surfactant, and stored thepreparations under normal vector storage conditions and, to acceleratethe deleterious effect of surfactant, at cycled elevated/cooledtemperature (a common drug stability technique called “accelerated”stability testing).

BRIEF SUMMARY

We found that, directly contrary to our own expectations and theteachings of the art, surfactant does not reduce vector infectivity. Tothe contrary, we found that surfactant preserves vector infectivity:vector mixed with surfactant after storage showed higher infectivitythan vector not mixed with surfactant. Further, surfactant provided aqualitatively-unexpected benefit, protecting viral vector from damagedue to transient elevated temperature.

Our discovery thus for the first time opens the door to several advancesin medical treatments. First, we have found a way to protect virusagainst heat damage by protecting the virus with surfactant. Thisenables therapeutic virus (e.g., vaccines or gene therapy vectors) to bemanufactured, shipped, stored and distributed at higher temperaturesthan previously thought possible. This lessens the expense ofdistribution and increases the scope of the area where the virus may besafely distributed to physicians and patients.

Second, our discovery enables virus manufacturers to increase the shelflife of viral treatments. This enables manufacturers to manufacture inlarger batch sizes, and may reduce the amount of virus-based medicineswhich must be discarded as being out-of-date.

BRIEF DESCRIPTION OF THE FIGURES

None

DETAILED DESCRIPTION

We tested the effect of surfactant on virus infectivity and surprisinglyfound that storage of a virus with surfactant does not in fact impairvirus infectivity. To the contrary, our results show surfactantpreserves it. This may be seen by our results in several experimentalExamples.

Our Examples use The rAd-IFN virus. The rAd-IFN virus is anon-replicating recombinant adenovirus type 5 (Ad5)-based, interferonalpha-2b (IFNα2b) gene transfer vector. It is being developed as e.g.,an intravesical treatment for superficial bladder cancer. rAd-IFN isused to deliver the human IFNα2b gene into cells in the urotheliallining of the bladder. The art teaches that on administration, Syn3™excipient is included in the Admixture formulation immediately beforeadministration to enhance gene transfer into bladder epithelium. Werefer to “Admixture” as the final formulation of rAd-IFN in a patientfinished dosage form; it contains rAd-IFN vector, Syn3 and finalformulation buffer (“FFB”).

Our discussion of these Examples use the following abbreviations:

-   Admixture A mixture of rAd-IFN and Syn3 and FFB representing the    patient dose in Phase III clinical trial of the gene therapy product-   DP Drug Product-   DS Drug Substance-   FFB Final Formulation Buffer, i.e., a sterile buffer suitable for    injection-   IFNα2b interferon alpha-2b-   rAd-IFN A replication deficient recombinant adenovirus type 5 based    interferon alpha-2b gene transfer vector-   RT Room temperature, ˜20 C.-   Syn3 SYN 3 is the polyamide surfactant commercially available from    the Schering-Plought Inc. division of Merck & Co. (Rahway, N.J.) and    having the structure shown:

-   -   The art also uses the abbreviation “SYN3” to refer to the        synapsin-3 protein, a protein involved in synaptic        neurotransmission. We here use the term to refer to the        synthetic surfactant, not the naturally-expressed        neurotransmitter polypeptide.

-   TS Test sample

Example 15-0029

The aim of this study was to assess the effect of Syn3 DP on potency ofthe rAd-IFN gene therapy product in rAd-IFN potency assay. At the sametime, two storage temperatures (RT and 2-8° C.) of Syn3 DP powder werecompared.

We used two samples of Syn3, one stored in RT and one at 2-8° C. Thisstudy entailed performing of two rAd-IFN potency assays, each has one TSmade from admixture, and a reference standard rAd-IFN from a knownquantified batch as a control sample. One assay has the admixture withRT stored Syn3 DP, the other admixture with Syn3 DP stored in thefridge. The potency assay was conducted as known in the art.

We prepared two admixtures made from these samples, and compared them toeach other and to reference standard material in a potency assay.Admixture containing 3×10¹¹ vp/ml of rAd-IFN was made and analyzed in astandard rAd-IFN potency assay. Two assays were run, both having ARKz001as a control sample/reference standard.

Materials and Methods:

We prepared the Admixture as follows:

a) Add 20 ml of sterile water using a 20 ml syringe and 21G needle toone vial of Syn3 DP sterile powder and gently agitate vial to mix for acouple of minutes. Allow contents to dissolve and bubbles to disappearfor 40 minutes at ambient temperature and light. After reconstitutionSyn3 DP should be stored at room temperature and must be used within 24hours. In this study, potency assay is started immediately after samplepreparation.

b) Take a vial of rAd-IFN DP (noting the batch number) from ultra-lowtemperature freezer (−70 C) and thaw at ambient temperature and lightwith periodic gentle agitation to mix contents while thawing. Oncethawed, invert each vial several times to ensure contents are thoroughlymixed.

c) Pipette 2.1 ml of FFB into a 15 ml tube.

d) Withdraw dissolved Syn3 DP using a 10 ml syringe and 21 G needle intoa separate container. Pipette 1.4 ml of Syn3 DP to the tube with FFB andinvert several times to mix contents.

e) Withdraw the rAd-IFN DP from the vial using 10 ml syringe and alonger 21 G needle. Use a separate container for the virus and pipette5.3 ml to the admixture tube.

f) Mix well by inverting admixture container several times.

g) Final vp/ml concentration was 3×1011 vp/ml and volume was 8.8 ml.

We then performed the rAd-IFN potency assay. The prepared Admixture(from above) was used as a test sample. To continue to virus dilutionsin potency assay, the admixture was diluted 1:6 in GM (1 ml ofadmixture+5 ml of GM; 5×1010 vp/ml), and then used as normal TS inpotency assay. The reference standard material (ARKz001) is treatednormally as in a standard procedure. To properly censor the resultingdata, before the tests were run, we decided that in case the assays didnot have enough passed plates/TS, the results would be given forinformation only.

Results from these first two analyses are summarized in the followingTable. The rAd-IFN in admixture has average relative potency of 1.29U/vp. Average potency value for rAd-IFN from three assays is 1.28 U/vp.

Potency results of admixtures with differently stored Syn3.

U/vp of U/vp of Assay Syn3 stored at admixture ARKz001 POT-15-037 RT1.24 1.06 POT-15-038 2-8° C. 1.34 0.94* Average 1.29 *Only one platepassed statistical testing, potency value here for information only.

Our results confirm that storage of Syn3 surfactant at RT does not infact adversely affect Syn3 by reducing the Potency of admixture madewith it; to the contrary, even after storage at RT, Syn3 appears toproduce acceptable Potency.

In this first Example, two admixtures containing rAd-IFN were used astest samples in two potency assays. The admixtures were prepared withSyn3 powder stored at RT and in the refrigerator to see if the storagecondition of the Syn3 powder might change the potency of the finalAdmixture. Our data from these assays served as proof that admixture assample matrix can in fact be analyzed in a standard Potency assay, i.e.,that surfactant does not interfere with a Potency assay.

From these first results, our conclusion is that admixture is a suitablesample type for potency assay and that storage in RT or fridge (2-8° C.)are both suitable for Syn3 powder as far as potency of the finaladmixture is concerned.

Example 15-0029-ADD1

An in-use stability study for Admixture has been done where stability ofadmixture in four different catheters up to 24 hours has been tested.Surplus admixture at time point zero was frozen as 128×500 μl inultra-low freezer (below −60° C.). Set of samples handled with avent-needle is chosen for this study.

Assays used to estimate the stability of stored admixture areinfectivity, potency, HPLC, pH and particle size. Total viral particleamount has been analyzed with HPLC instead of OD₂₆₀.

ARKz001 was employed as the control/reference standard sample in bothassays and its potency values were within typically seen values. In onepotency test, ARKz001 only passed statistical equivalence testing withone plate, so the potency value here is reported for information only.Nevertheless, the potency values from all of the plates were withinexpected potency values for ARKz001.

The aim of this follow-up work is to provide a baseline measures for astudy of stability of rAd-IFN in its “ready-to-use” clinical formulation(as “Admixture”) with diluent (sterile injectable buffer) and surfactant(e.g., Syn3™) when the preparation is thawed and frozen several timesover a period of time per Example 15-0035 below.

Sixty-four (64) 500 μl aliquots of vent-needle admixture (prepared foradditional testing) will be reserved as test samples in this work. Allaliquots are thawed at each time point and three or four vials are takenfor analyses. (1 vial for HPLC, 1 vial for particle size, 1 vial forinfective titer and potency, 1 vial for pH) All remaining vials arefrozen again to below −60° C. All analyses should be done on the sameday if possible. If repeat assays are needed, individual vials will bethawed as needed.

Control Samples: Half of the aliquots (64 pcs) as prepared earlier (ventneedle, additional testing) are reserved as control samples. Thesesamples are thawed only as needed for each assay, so they will have onlyone freeze-thaw cycle before analyzing.

Analytical Methods: The analyses in this and Example 15-0035 will bedone as presented in this Table. The time point windows are in −AT1.

Stability study set-up Time Infective Particle point HPLC titre Potencysize pH 0 M x x x x x 3 M x x x x NA 6 M x x x x NA 9 M x x x x NA 12 Mx x x x x

Thawing Test Samples: The procedure to thaw test samples will be asfollows:

(1) At the first time point, take sixty-four (64) vials (test samples)of vent-needle admixture and put them into a separate storage containerfrom the other 64 vials, which are reserved for control samples.Continue with steps a)-d).

(2) At other time points go straight to step a).

-   a) Take all remaining aliquots that are intended for test samples    and thaw them at RT until they melt.-   b) Take three aliquots and put them in the fridge until assays can    be done (on the same day).-   c) Put the rest of the vials back to the ultra-low freezer.-   d) Mark down in −AT1 of this plan that the aliquots have been thawed    and frozen. Keep the attachment together with sample submission    form/ultra-low freezer content logbook for the samples.    Testing is continued up to a year, which means that the test samples    will undergo up to four thaws during this study whereas control    samples only one.

HPLC is done on the samples, with a concentration of admixture ofapproximately 3×10¹¹ vp/ml. Infectivity of admixture is determined withflow cytometry; test sample together with the control/reference standardsample are measured in the same assay. Particle size analysis is doneaccording to the dynamic light scattering method. The pH is determinedat the last time point, to see if repeated thawing and freezing hasaffected e.g. microbial growth in the vials.

The results from the assays done at each time point are collected attime=0, 3, 6, 9 and 12 months, along with assays running numbers. Thereis a window of ±two weeks for the analyses. This study is continued upto 12 months. The results of the assays are compared to time point zeroand to the control/reference standard sample that is run at every timepoint but with less freeze-thaw cycles. The time point zero resultshould be identical for both the test samples and the control/referencestandard sample.

Our results for time t=0 are as follows:

Results for 15-0029-ADD1 Viral Viral % % % Particle Particle changechange change concen- Concen- from from from Appear- tration trationprevious Infectivity previous previous Particle ance pH (O.D.260) (HPLC)measure Assay measure Potency measure Size Opalescent 7.76 10¹² 3.72¹¹N/A 3.73¹⁰ N/A 1.13 U/vp N/A 113 colorless vp/mL vp/mL NASIU/ (forinfor- nm solution, (for mL mation no infor- only) visible mationparticles only) Time = 0 These data confirm that the samples at t = 0have been properly prepared.

Example 15-0035

The aim of this study is to provide in-use stability andbiocompatibility data for rAd-IFN in Admixture. From the samplesprepared in Example 15-0015 ADD1 (above), sixty-four (64) vials havebeen be reserved as control samples, which are only thawed as needed forassays upon each time point. The rest of the vials will undergo severalsequential freeze-thaw steps as they are all thawed at each time point,the needed vials are removed for analysis and the rest are frozen back.

rAd-IFN is a replication deficient recombinant adenovirus type 5 (rAd5)based gene transfer vector containing human interferon alpha-2b (IFN)gene in expression cassette that replaces E1a, E1b and pIX regions atthe 5′ end of adenovirus genome. The vector has been developed forintravesical treatment of non-muscle invasive bladder cancer. It iscurrently heading towards Phase III clinical trial. Formerly the productwas known by the code SCH 721015 but currently it is referred asrAd-IFN.

rAd-IFN vector is used to deliver the human IFNα2b gene into cells inthe urothelial lining of the bladder. Before intravesicaladministration, Admixture is prepared containing appropriate dose of therAd-IFN drug product in final formulation buffer (FFB) and 1 mg/ml Syn3excipient which is included in the Admixture formulation to enhance genetransfer into bladder epithelium. This plan describes in-use stabilityand biocompatibility study protocol for rAd-IFN in admixture bymimicking phase III clinical scenario. The Admixture containing 3×10¹¹vp/ml (Dose level 2) is exposed various specified delivery componentsduring admixture preparation followed by holding in IntraVia sterilebag, withdrawal into syringes with 1 hour incubation and catheteradministration. The study will include sampling at three different timepoints with four different catheter types:

-   -   Zero time point=Preparation of Admixture into IntraVia sterile        bag followed by withdrawal into syringe, and delivery via four        different types of catheters.    -   Hold 8 h/RT time point=Admixture preparation into IntraVia        sterile bag and holding in the dark at room temperature (RT) for        8 hours followed by 1 hour incubation at RT and ambient light,        withdrawal into syringes followed by 1 hour incubation at RT and        ambient light and delivery via four different types of        catheters.    -   Hold 24 h/+4° C. time point=Admixture preparation into IntraVia        sterile bag and holding at refrigerated conditions (2-8° C.) for        24 hours followed by 1 hour incubation at RT and ambient light,        withdrawal into syringes followed by 1 hour incubation at RT and        ambient light and delivery via four different types of        catheters.        The stability and biocompatibility will be assessed using the        following assays to characterize any changes in appearance, the        physical and chemical characteristics, biological properties and        virus particle concentration and particle size of rAd-IFN in        Admixture:    -   pH (Ph. Eur. 2.2.3)    -   Appearance (USP chapter 790 and Ph. Eur. 2.2.1 visual method,        and Ph:Eur, 2.2.2 modification of method I)    -   Potency assay    -   Infectivity assay    -   HPLC    -   OD₂₆₀    -   Particle size

The admixture components are:

rAd-IFN DP—rAd-IFN-150702, 4.86×10¹¹ vp/ml, stored at Ultra low freezer

Syn3 DP—Lyophilised powder lot 14071, stored at 2-8° C.

rAD-IFN FFB—rAd-IFN Diluent buffer lot FVTc001, stored at 2-8° C.

Aqua ad Iniectabilia

Schedule for the in-use stability and biocompatibility study ispresented in Table II. The prepared Admixture will be divided equallyinto two 60 ml syringes and will be used for administration of twodifferent catheters. Both catheters are operated at same time by twoseparate operators. There are no hold step for zero time point, transfervia catheters and sampling will be performed immediately after admixturepreparation followed by sample analysis. For the Hold 8 h/RT time point,prepared Admixture in IntraVia bag will be stored in the dark for 8hours at RT. For the Hold 24 h/+4° C. time point, prepared Admixture inIntraVia bag will be stored for 24 hours at fridge. Both hold timepoints also include 1 hour incubation in IntraVia bag at RT and ambientlight before withdrawal into 60 ml syringes and 1 hour incubation at RTand ambient light in syringes.

TABLE II Schedule for the in-use stability and biocompatibility studyWeek −2 Week −2 Week 1 Week 1 Week 2 Week 2 Day 1 Day 2 Day 1 Day 2 Day1 Day 2 Zero Admixture Admixture time preparation, preparation, pointCat1 and Cat3 and Cat2 Cat4 sampling sampling and and analysis analysisHold Admixture Admixture 8 h/RT preparation, preparation, time Cat1 andCat3 and point Cat2 Cat4 sampling sampling and and analysis analysisHold Admixture Cat1 and Admixture Cat3 and 24 h/ preparation Cat2preparation, Cat4 +4° C. sampling sampling time and and point analysisanalysis

Preparation of Admixture:

The Admixture containing 3×10¹¹ vp/ml (Dose level 2) may be preparedunder laminar flow hood as follows:

-   -   1. To obtain a Syn3 solution, add sterile water using a syringe        and needle to a vial of Syn3 powder and gently agitate vial        to mix. Allow contents to dissolve. After reconstitution Syn3        should be used within 24 hours.    -   2. Remove vials of rAd-IFN from the freezer and thaw with        periodic gentle agitation. Once thawed, invert each vial several        times to ensure contents are thoroughly mixed.    -   3. Add Dilution buffer into an empty mixing vessel.    -   4. Withdraw dissolved Syn3 solution and introduce into mixing        vessel containing the Dilution buffer. Invert the vessel several        times to mix contents well.    -   5. Add rAd-IFN to the mixing vessel and mix gently.        We intend to use the following Analytical assays:

Biological activity of rAd-IFN will be assessed by determining potencyand infectivity in suitable cell lines. Appearance will be determinedand physical and chemical properties will be characterized by measuringpH. Viral particle concentration and size will be determined using highperformance liquid chromatography (HPLC), absorbance at 260 nm (OD260)and particle size assays. All methods are qualified/assay qualificationis on-going for this product.

After the completion of the single assay the accepted result will betransferred to a result collection form or laboratory notebook whichwill contain all the results and assay references for this stabilitystudy.

Potency: The potency assay models all steps of the therapeutic mechanismof rAD-IFN: infection of cells, expression of IFN and antitumor activityof the product and measures the killing efficacy of rAd-IFN. Interferonsensitive human bladder cancer cells are transduced using multipledilutions of reference standard and test samples leading to expressionof IFNα2b and subsequent cell death. Interferon-sensitive cells may bepurchased commercially from several sources, or may be readily preparedor isolated as is well known in the art. See e.g., Howard R. Hubbell etal., Independent sensitivity of Human Tumor Cell Lines To Interferon andDouble-Stranded RNA, 44 Cancer Res., 3252 (1984); Lawrence M. Pfeffer etal., Cytoskeletal Association of Human a-Interferon Receptor ComplexesIn Interferon-Sensitive and -Resistant Lymphoblastoid Cells, 84 P.N.A.S.3249, 3249 col. 1 (1987). Cell killing efficiency is determined using acolorimetric method measuring dehydrogenase activity of the livingcells. The relative potency of the test sample is determined against thereference standard response curve after testing parallelism byequivalence test as outlined in Ph. Eur. 5.3 and USP <1034>. The assaymay be performed as described in Viral Vector Assay and Vector, U.S.Patent Application Ser. No. 62/218,810 filed 15 Sep. 2015, incorporatedhere by reference.

Infectivity: In infectivity assay, cells that support adenovirusreplication are infected with different concentrations of adenovirus.After infection, percentage of infected cells is determined with a flowcytometer utilizing a fluorescently conjugated antibody against anadenoviral structural protein. Samples are analysed in parallel with areference standard and infectivity is given as relative InfectiousUnits/ml. Result is calculated using Slope Ratio method as outlined inPh. Eur. 5.3 and USP <1034> The assay may be performed as described inViral Vector Assay and Vector, U.S. Patent Application Ser. No.62/218,810 filed 15 Sep. 2015.

pH: pH of rAd-IFN drug products is buffered around pH 7.8 to ensureacceptability for instillation and long term maintenance of theadenovirus function. The desired pH is confirmed by measuring the samplein solution using a standardized pH meter at room temperature. Test isdone according to Ph. Eur. 2.2.3 and USP <38>.

Appearance: Appearance (clarity and degree of opalescence, degree ofcoloration and determination of visible particulates) of adenoviralvector samples is determined according to European Pharmacopoeia (EP)using visual method (EP 2.2.1 visual method and EP 2.2.2 modification ofmethod I) harmonized with United States Pharmacopoeia (USP) chapter 790guidelines. The assay may be performed as described in Viral VectorAssay and Vector, U.S. Patent Application Ser. No. 62/218,810 filed 15Sep. 2015.

HPC: HPLC is used to determine total viral particle of rAd-IFN. Workingstandard (WS) is used to establish a standard curve. The obtained peakareas are plotted against known virus particle concentration of WS(vp/ml). Virus particle concentration of test samples is interpolatedfrom the standard curve and multiplied with dilution factor. Testsamples are analysed as duplicates, and results are reported as averageresult of two replicate samples. The assay may be performed as describedin Viral Vector Assay and Vector, U.S. Patent Application Ser. No.62/218,810 filed 15 Sep. 2015.

OD₂₆₀: Total viral particle concentration of a drug product must reflectthe safe and efficacious dose. The concentration of rAd-IFN isdetermined spectrophotometrically by measuring optical density at 260 nm(OD₂₆₀) in presence of 0.1% SDS. SDS is used to lyse the viral capsid. Aconversion factor of 1.1×10¹² particles per absorbance unit at 260 nm isused. This value is based on scientific literature as well as internalfindings. Negative control is used to determine background absorbance tobe subtracted from the results and positive control to confirm the assayperformance.

Particle size: Particle size assay is used to monitor possible particleaggregation. The assay is based on dynamic light scattering, where alaser beam is directed to the analyzed sample and the intensity ofscattered light is measured from specific detection angle. The smallerthe size of soluble particles, the faster the particles movement in thesurrounding liquid and the faster the intensity changes of scatteredlight. Particle size can be calculated based on data obtained from thefluctuations in the light intensity versus time profile. Particle sizeassay for rAd-IFN may be performed as known in the art.

Summary

Given our examples, one can derive from them a number of therapeuticallyvaluable and commercially valuable applications. These include, forexample:

-   -   1. A composition of matter comprising an infective virus in a        therapeutically-effective amount and lyophilized to remove        substantially all water, mixed with a surfactant in an amount        effective to preserve the infectivity of the virus.    -   2. The composition of matter of paragraph 1, wherein the        surfactant is in an amount effective to preserve the infectivity        of the virus during storage at elevated temperature.    -   3. The composition of matter of paragraph 1, wherein the        surfactant comprises Syn3.    -   4. The composition of matter of matter of paragraph 1, wherein        the infective virus functions as a therapeutic vaccine.    -   5. The composition of matter of paragraph 1, wherein the        infective virus functions as a gene therapy vector.

Given our examples, the artisan may readily devise further variants andmodifications on our general theme. For example, while our actualexperimental examples involve Syn3, other surfactants are known in theart as useful for increasing viral transfection efficiency. Functionallyequivalent surfactants are described, for example, in William BENEDICT,Methods and Compositions for Treatment of Interferon Resistant Tumors,WO 2005/058368 (30 Jun. 2005) at [0054]-[0055].

Similarly, while our actual experiments used replication-deficientvirus, we expect our invention would provide the same benefit toreplication-competent virus. Similarly, while our actual experimentsused adenovirus, we expect our invention would provide similar benefitsto other infective virus preparations using other genera of virus (e.g.,adeno-associated virus) and other specific viruses (e.g., measles, mumpsand rubella attenuated live virus, oral polio, etc.). Indeed, we expectour invention to also be useful for attenuated bacterial vaccines whichuse bacterium which are live yet inactive (e.g., Rickettsia sp.,Salmonella typhi and Bacillus Calmette-Guérin vaccines).

Similarly, certain of our claims require “transient” storage at elevatedtemperature. We intend this term to cover periods longer than that knownin the art to not adversely affect virus vector infectivity, but notpermanent or long-term storage; longer than half an hour perhaps, butshorter than a week. We thus intend the coverage of our patent to bedefined not by our specific examples discussed here, but by the legalclaims and permissible legal equivalents of them.

1-10. (canceled)
 11. A method of preserving the infectivity of aninfective vector, comprising: mixing an infective agent with asurfactant with an amount effective to preserve the infectivity of theagent, removing substantially all water from the mixture, and storingthe mixture.
 12. The method of claim 11, where the infective vectorcomprises infective virus.
 13. The method of claim 12, where thesurfactant comprises NODA.
 14. A composition comprising an infectiverecombinant adenovirus and a surfactant, the surfactant in an amounteffective to preserve the infectivity of the recombinant adenovirusagent, whereby (a) the infectivity of the recombinant adenoviruscombined with the surfactant is at least 100% of (b) the infectivity ofthe recombinant adenovirus without the surfactant when stored under thesame conditions and for the same time, and wherein the surfactant hasStructure I:


15. The composition of claim 14, wherein (a) the infectivity of therecombinant adenovirus combined with the surfactant is at least 117% of(b) the infectivity of the recombinant adenovirus without the surfactantwhen stored under the same conditions and for the same time.
 16. Thecomposition of claim 14, wherein (a) the infectivity of the recombinantadenovirus combined with the surfactant is at least 129% of (b) theinfectivity of the recombinant adenovirus without the surfactant whenstored under the same conditions and for the same time.
 17. Thecomposition of claim 14, wherein (a) the infectivity of the recombinantadenovirus combined with the surfactant is at least 142% of (b) theinfectivity of the recombinant adenovirus without the surfactant whenstored under the same conditions and for the same time.
 18. Thecomposition of claim 14, whereby (a) the infectivity of the recombinantadenovirus combined with the surfactant is at least 100% of (b) theinfectivity of the recombinant adenovirus without the surfactant whenstored under the same conditions for at least three months.
 19. Thecomposition of claim 18, whereby (a) the infectivity of the recombinantadenovirus combined with the surfactant is at least 100% of (b) theinfectivity of the recombinant adenovirus without the surfactant whenstored under the same conditions for twelve months.
 20. The compositionof claim 14, whereby (a) the infectivity of the recombinant adenoviruscombined with the surfactant is at least 100% of (b) the infectivity ofthe recombinant adenovirus without the surfactant when stored under thesame conditions and for the same time, wherein the conditions compriseat least one freeze-thaw cycle. 21.-32. (canceled)
 33. The compositionof claim 14, wherein the recombinant adenovirus isreplication-deficient.
 34. The composition of claim 14, wherein thesurfactant is in an amount effective to preserve the infectivity of therecombinant adenovirus at a temperature of 2° C. 35.-36. (canceled) 37.The composition of claim 14, wherein the recombinant adenovirusfunctions as a therapeutic vaccine.
 38. The composition of claim 14,wherein the recombinant adenovirus functions as a gene therapy vector.39. The composition of claim 38, wherein the recombinant adenovirus isreplication-deficient and cannot replicate in normal human cells. 40.The composition of claim 43, wherein the composition comprisesnadofaragene firadenovec.
 41. The composition of claim 14, wherein theinfective recombinant adenovirus comprises a transgene.
 42. Thecomposition of claim 41, wherein the transgene encodes interferon. 43.The composition of claim 42, wherein the interferon is interferonalpha-2b.